Cryptococcus neoformans is an opportunistic fungal pathogen that has significant clinical impact on immunocompromised patients. In particular, patients with AIDS are exquisitely vulnerable to cryptococcosis. The most common clinical presentations are pulmonary cryptococcosis and meningoencephalitis. Current antifungal agents are inadequate for safe and effective therapy. Over the past decade, the pathogenesis of C. neoformans has been intensively studied using molecular biology, genetic, immunological and biochemical approaches. Functional genomics has the potential to accelerate our understanding of pathogenesis in complex organisms. Because of two recent advances, the opportunity exists to efficiently test the contribution of C. neoformans genes to pathogenesis using a mass screening approach. First, we have developed a genetic screen for C. neoformans that allows identification of virulence mutants from a large pool of insertion mutants. This screen increases the efficiency and rapidity of identification of genes which alter the pathogenicity and eliminates the need to test each and every mutant individually in an animal model - thus reducing cost, minimizing the use of animals, and accelerating results. Second, a genome project with the goal of sequencing the entire genome of C. neoformans is currently underway. This project will provide the raw data for identification of the vast majority of potential open reading frames in C. neoformans. Each open reading frame is potentially important for pathogenesis. We propose to develop technology to rapidly construct targeted insertions in C. neoformans open reading frames and estimate that we can disrupt approximately 6 percent of the open reading frames from C. neoformans. This project will focus on genes important for biogenesis and maintenance of the cell wall. Each insertion will be tagged with a unique sequence that will allow identification of that mutant within a large pool of mutants. Large groups of mutants will be screened for growth in a mouse model, and mutations in genes that affect virulence in this competitive assay will be rapidly identified. The bank of specific insertional mutants that we will generate for this project represents a valuable resource for the development of novel antifungal targets studies of pathogenesis and host response.